1. The spleen, not the lung, is the major organ where T-cells acquire pathogenicity This study, dealing with identification of the organ where T-cells acquire pathogenicity, was initiated in FY 2013 and has been extended in FY 2014. In the experimental system used for this ongoing study, mouse T-cells specific against hen egg lysozyme (HEL) are transferred into mice in which HEL is transgenically expressed in the eyes. When activated, the transferred cells induce ocular inflammation, that is detected after a latent period of 2-3 days post cell transfer. This delay of disease onset is attributed to the need for a period of licensing for pathogenicity during which the transferred cells acquire the capacity to induce inflammation. Our previous data indicated that the majority of transferred cells accumulate in the spleen before invading the eye. This observation contradicts the conclusion of a Nature paper (Odoardi et al., 2012, 488:675), claiming that the lung is the licensing organ for pathogenic T-lymphocytes. Data we collected in 2014 have provided evidence to support our assertion that the spleen, not the lung, is the major licensing organ. To rule out the possibility that the donor cells in the spleen were licensed in the lung prior to homing to the spleen, we showed that the donor cells in the spleen resemble those in the lung in their pattern of changes in their cell markers during the licensing period. The tested markers are related to the acquisition of the capacity to migrate and invade non-lymphoid organs. The similarities were determined by both immuostaining and microarray technologies. We immunostained the donor cells in the recipients lung and spleen collected on days 1, 2, 3, 4 and 7 post cell transfer. A correlation was found between the donor cells in the lung and spleen in their temporal changes for all tested markers, that included CCR1, CCR3, CCR5, CCR7, CXCR3. alpha4beta7, alphaEbeta7 and S1P1. The microarray analysis was carried out by the Nanostring method in which the expression of 500 genes related to inflammation are analyzed. Data collected by this microarray assay are in accord with those obtained by the immunostaining: we found similar patterns of increase or decrease in expression of genes of molecules involved in inflammation induction by the donor cells in the recipients lung and spleen on days 2, 3, 4 and 7 post cell transfer. Together, the findings with the two methods indicate that donor cells homing to the spleen resemble those homing to the lung in their patterns of undergoing biochemical changes (licensing for pathogenicity), that enable them initiate inflammatory responses in the target organ. These findings thus rule out the possibility that donor cells residing in the recipient spleen were previously licensed in the lung. Since the number of the donor cells in the spleen is larger by far than that in the lung ( X10), our data thus show that the conclusion reached by Odoardi et al in their Nature paper, that the lung is the site of licensing for pathogenicity, is incorrect: the spleen is the major site for this process! 2. Toxic effects of digoxin on the retina This study, initiated in FY 2013, is dealing with the immunosuppressive and toxic activities of digoxin in mice developing EAU and has been extended in FY 2014. Digoxin is a well known medication for certain heart diseases and has been recently reported to be capable of inhibiting experimental autoimmune diseases in mice (Huh et al., Nature, 2011, 472:486). We found that digoxin also inhibited EAU development, but, in addition, it is toxic to the retina. New findings include: (i) Different strains of mice vary in their susceptibility to the toxic activity of digoxin, demonstrated by loss of the photoreceptor cells and thinning of the retina. Thus, retinae of B10.A mice treated with digoxin were remarkably more affected by the toxic effect than those of mice of the (FVB/N x B10.BR)F1 line. (ii) The retinal damage in digoxin-treated mice was also assesssed by electroretinographic (ERG) analysis. The treatment effect was found to be dose-dependent and was depicted by lowered peak potentials of both the a-wave and the b-wave potentials. Of interest was also the finding that the loss of visual function plateaued after day 7 of the daily treatment, with little change in visual function seen between days 7 and 14 of treatment. Our findings thus indicate that digoxin should be avoided as a treatment of heart disease because of its toxicity to the retina.